The present invention relates generally to the culture and preservation of cells, and more specifically to the culture and preservation of mammalian cells using the ovarian fluid produced by spawning fish. The method has significant advantages over conventional methods, which use whole serum or plasma components derived from mammals or fish.
The culture of animal cells requires a defined medium containing a specific quantity of certain chemicals, and for the culture of most mammalian cells, an undefined nutrient supplement (medium) is also required. From the earliest attempts at cell culture to the present, this nutrient medium has been derived exclusively from animal or human blood. Fetal bovine serum (FBS) remains the most common nutrient medium used for the culture of animal cells. However, in order to eliminate certain risks, and to better quantify media components, a recent trend is to use so-called xe2x80x9cserum-freexe2x80x9d media. These formulations are not truly serum-free, but rather contain specific quantities of certain serum or plasma components, such as albumin or lipids, in place of whole serum.
This reliance on the use of blood components as nutrients in mammalian cell culture has led to an increased concern over the presence of possible infectious agents in the cells. For example, problems remain in achieving 100% viral inactivation in blood products without compromising quality. An equally serious concern is the emergence of transmissable spongiform encephalopathies (TSEs) in mammalian blood and plasma components. The latter problem is especially difficult to overcome, since at present it is not possible to predict which donors, animal or human, will later develop a prion disease. Although some plasma proteins can be produced by recombinant technology, others, such as many glycoproteins, cannot. In addition, recombinant proteins are generally very expensive.
In order to reduce the risks from mammalian infectious agents, a whole serum product has been developed from fish, particularly salmon and trout, for mammalian and insect cell culture. Although this material is a satisfactory substitute for FBS in a few cell lines, its usefulness is limited by high levels of non-protein nitrogen (NPN), by large amounts of lipids that are easily oxidized, and especially, by proteases that are active at 4xc2x0 C. The latter problem contraindicates the use of salmonid serum as a medium for preservation and storage of cells at low temperature.
In addition to fish whole serum for mammalian cell culture, fish plasma components, specifically antifreeze glycoproteins (AFGPs) from polar fishes, have been used successfully in low temperature preservation of cells such as human platelets. Although these AFGPs appear promising, their availability is severely limited by the relatively small numbers and size of polar fishes, their natural source, and the difficulty of producing the recombinant glycoprotein.
There is therefore a great need for a nutrient medium that overcomes or minimizes the foregoing problems, and which is also practical for widespread use.
While the field of cell culture has recently come to include blood products from fish, the ovarian fluid of fish has been of interest only to those in fishery science. The ovary of salmonid fishes lacks an ovarian membrane, and ovulated eggs are free in the body cavity surrounded by the slightly viscous fluid. At spawning, the eggs and ovarian fluid are expelled through the genital pore. Ovarian fluid has been studied for its role in salmonid reproduction and for its chemical composition, its novel proteins, and its utility in testing for the presence of fish disease.
According to the present invention, this ovarian fluid produced by spawning female salmon or trout is used to replace whole serum or plasma components as a nutrient medium in mammalian cell culture. The use of this substance as a nutrient medium in the place of whole serum or plasma components promotes the growth and proliferation of the mammalian cells (for example, NIH 3T3 and M2 cells). Use of fish ovarian fluid eliminates the risks and uncertainties inherent in the use of mammalian blood products, as well as the limiting factors of NPN, lipids, and proteases associated with fish serum or plasma when used as nutrient media.
Further, according to the present invention, salmonid ovarian fluid is used in low-temperature preservation of mammalian cells, especially human platelets. With present technology, these cells must be stored at or above 22xc2x0 C., as lower temperatures produce xe2x80x9cactivationxe2x80x9d, that is, a process marked by aggregation and change in shape, and resulting in irreversible cellular damage. Storage at 4xc2x0 C. could dramatically increase the useful life of the mammalian cells, now limited to less than a week at 22xc2x0 C.
Several factors have hindered, or have led those of skill in the art away from, the previous use of fish ovarian fluid for mammalian cell culture and preservation. For example, the field of cell culture and storage, including platelet preservation, is distinctly different from the field of fishery science; those skilled in working with mammalian cells would be unlikely to know of the existence of ovarian fluid and therefore could not speculate on its utility. Certainly, there has been no suggestion to motivate those working with mammalian cells to expend the time, effort, and expense necessary to experiment with fish ovarian fluid to culture or preserve mammalian cells.
Further, utilization of blood products as the nutrient supplements in mammalian cell culture is firmly established. Use of a nutrient fluid from a source other than blood would appear unlikely to be advantageous to one skilled in the art of animal cell culture, particularly absent of any suggestion by those in the field of an advantage.
In addition, the presence of ovarian fluid in wild fish is relatively brief and seasonal, unlike that of blood. Also, spawning time in wild fish under natural conditions varies among stocks and individual fish. These and other factors make it logistically difficult to obtain practically usable quantities of ovarian fluid from wild fish.
Salmonid ovarian fluid contains very small amounts of the components generally considered essential for cell growth, and therefore would appear to be a poor choice for a nutrient medium. For example, the standard nutrient medium FSB contains about 4 g/dL protein and 40 mg/dL cholesterol. Similarly, salmonid serum contains about 4 g/dL protein and more than 200 mg/dL cholesterol. A representative sample of pooled ovarian fluid has been analyzed and was found to contain only 0.2 g/dL total protein and 6 mg/dL cholesterol. It also has been found that ovarian fluid from chum salmon contained only about 10% of the protein and cholesterol found in the plasma. Known and hypothesized functions of ovarian fluid include protection of the eggs from physical or osmotic damage, and maintenance of sperm motility. These functions do not suggest a role for ovarian fluid in mammalian cell culture or preservation.
The use of blood products from the coldwater fishes for mammalian cell culture or storage offers safety from mammalian infectious agents. However, and despite the indications otherwise, it has been found in the extensive research leading to the development of the present invention that the use of ovarian fluid from these fish provides significant advantages over the use of fish blood products.
For example, analyses of salmonid ovarian fluid show very low levels of NPN, lipids and protease activity, factors that have proven inhibitory in the use of fish serum for culture or preservation of mammalian cells.
Further, Salmonid ovarian fluid contains protease inhibitors. As a result of the research, it has been determined that trout ovarian fluid contains a thrombin inhibitor, and anti-proteolytic activity has been observed in fish ovarian fluid.
In addition, ovarian fluid can be obtained from farmed salmonids in at least twice the quantity of serum. Only about 50 ml of serum (100 mls of whole blood) can be obtained from even the largest (5-6 kg) farmed salmon, but a 2-4 kg fish can produce 100 mls of ovarian fluid. This large volume of ovarian fluid was also noted in trout. Farmed fish also provide ovarian fluid that is more consistent than that of wild fish, leading to more reproducible results.
As part of the research it was also found that salmonid ovarian fluid contains components similar to those involved in critical cell functions, but not identical to those provided by animal serum and plasma components. The polysialic acids are involved in organogenesis and cell adhesion and growth. Salmonid ovarian fluid contains a polysialic acid, deaminated neuraminic acid (KDN). This neuraminic acid is found in a KDN-rich glycoprotein and KDN-containing glycosphingolipids of trout ovarian fluid.
Thus, according to an aspect of the present invention, a method of using fish ovarian fluid for culture and preservation of mammalian cells includes obtaining ovarian fluid from a fish; culturing mammalian cells in first media including a first commercially defined medium and a first nutrient medium; removing the mammalian cells from the first media; replating the mammalian cells in second media including a second commercially defined medium and a second nutrient medium including the fish ovarian fluid; and continuing to culture the cells.
According to another aspect of the present invention, a method of using fish ovarian fluid for culture and preservation of mammalian cells includes obtaining ovarian fluid from a fish, and culturing mammalian cells in media including a commercially defined medium and a nutrient medium, wherein the nutrient medium includes the fish ovarian fluid.
According to still another aspect of the present invention, a method of preserving living cells includes obtaining ovarian fluid from a fish, isolating the living cells, and adding the fish ovarian fluid to the living cells.